1. Field of the Invention
This invention relates generally to methods of identifying bioactive small molecules from bacteria, fungi, and other microbes and more specifically to bioactive antibiotics, anti-fungals, and anthelmintics.
2. Background Information
Bacteria belonging to the order Actinomycetales, in particular those of the genus Streptomyces, constitute the most important and prolific source of antibiotics for medical, veterinary, and agricultural use. Streptomyces spp. are filamentous, non-motile bacteria found predominantly in soil and marine sediment. Unlike most other bacteria, they have complex secondary metabolic pathways that enable them to synthesize numerous structurally diverse molecules with a broad spectrum of bioactivity, especially antibiotics. Beginning with streptothricin and streptomycin in the early 1940s, the order Actinomycetales has yielded approximately 3000 known antibiotics. About 90% of these compounds were originally isolated from a Streptomyces spp. bacterium or are semi-synthetic derivatives of naturally-occurring molecules produced by a member of the Streptomyces (PMID: 11702082). Several notable examples include tetracycline (Streptomyces aureofaciens), chloramphenicol (Streptomyces venezuelae), vancomycin (Amycolatopsis orientalis), daptomycin (Streptomyces roseosporus), fosfomycin (Streptomyces fradiae), streptomycin (Streptomyces griseus) and erythromycin (Saccharopolyspora erythraea). A more extensive but not exhaustive list of antibiotics and the bacterium from which they were first isolated has been compiled in Practical Streptomyces Genetics (ISBN: 0708406238).
Although most famous for antibiotic production, Actinomycetes also produce compounds that have other useful properties in human and veterinary medicine as well as agriculture. Many anti-cancer agents, antifungals, anthelmintics, immunosuppressants, and other drugs currently in clinical use are derivatives of compounds that were originally isolated from an Actinomycete. This list includes bleomycin (Streptomyces verticillus; anti-cancer); nystatin (Streptomyces noursei; antifungal); amphotericin B (Streptomyces nodosus; antifungal); avermectin (Streptomyces avermitilis; anthelmintic); and rapamycin (Streptomyces hygroscopicus; immunosuppressant). Among a myriad of uses in agriculture, compounds isolated from Actinomycetes have been used as insecticides, herbicides, and to prolong the shelf-life of packaged foods. Specific examples from these areas are spinosyns (Saccharopolyspora spp.; insecticides) and natamycin (Streptomyces natalensis; food preservative).
Many bioactive compounds produced by Streptomyces spp., including antibiotics, fall into three main chemical classes: polyketides, non-ribosomal peptides, or hybrids of the two. As the name suggests, polyketides contain multiple ketone groups that are sometimes reduced to a lower oxidation state during various biosynthesis steps. This class of molecules is synthesized by polyketide synthases (PKS), a family of enzymes whose protein structure and corresponding genes are frequently organized in a modular structure. In turn, each module within the protein frequently contains several catalytic domains that have very specific functions. Three domains, thiolation, condensation, and adenylation, make up the core of each module. The coordinated action of each domain within each module leads to step-wise biosynthesis of polyketides that has been likened to an assembly line process. Tailoring enzymes then modify the polyketide, for example through glycosylation, oxidation, alkylation, and other chemical modifications, to generate the final structure. Non-ribosomal peptides are characterized by the presence of multiple contiguous amino acid residues within the molecule, for example β-lactams, vancomycin, and daptomycin, and are synthesized without the need for an mRNA template or the ribosome. As with PKS enzymes, non-ribosomal peptide synthases (NRPS) also have a modular organization, and many non-ribosomal peptides frequently undergo post-NRPS chemical modifications. The number of modules in PKS, NRPS and hybrid PKS/NRPS systems can vary over a wide range. Streptomyces albulus contains an NPRS cluster made up of only one module (PMID: 18997795), but more commonly there are several. These observations make clear that, fundamentally, the genome sequence of a producer organism defines the chemical structure of all polyketides, non-ribosomal peptides, and hybrid polyketide-non-ribosomal peptides it synthesizes.
The proven ability of Actinomycetes to produce clinically useful antibiotics, the deep knowledge acquired regarding their biology, and the existence of genetic manipulation tools for several species within this family of bacteria continue to make Actinomycetes an attractive source for new antibiotics. Indeed, Streptomyces coelicolor A3(2) (PMID: 12000953) and Streptomyces avermitilis (PMID: 12692562) were the first two members of this genus to be fully sequenced and found to have the capacity to produce many more secondary metabolites than had been isolated from either organism at the time. This pattern continues to hold even as the genomes from increasing numbers of Actinomycetes are fully sequenced (PMID: 17369815, 20624727, 18375553). Known, cultivable Actinomycetes consequently appear to harbor a large reservoir of potentially commercially-valuable bioactive compounds that still await discovery. In addition, it has been estimated that less than 1 part in 1012 of the earth's soil surface has been screened for Actinomycetes (Baltz, R. H. Antibiotic discovery from actinomycetes: will a renaissance follow the decline and fall? SIM News 55, 186-196 (2005)), a number that suggests the biosphere contains an even greater amount of undiscovered useful compounds.
These observations have spurred intense efforts to discover new bioactive molecules from Actinomycetes using a variety of methods. One common tactic is to search different parts of the world for new bacteria capable of producing secondary metabolites. Recent efforts focused on the marine environment in particular have led to the discovery of scores of new Actinomycetes (PMID: 12548698, 19406773, 19625431, 19196758, 19329599, 16538400). One of them, Salinospora tropica, was found to produce a compound, salinosporamide A, that exhibited potent and selective cytotoxicity against cancer cells. It has now advanced to clinical trials in humans for the treatment of multiple myeloma. Notwithstanding successes such as this one, high false positive rates plague bio-prospecting because the most abundant antibiotics in nature appear to be those that have already been discovered, a circumstance that interferes significantly with the screening process. For example, about 1% of soil actinomycetes produce streptomycin, first discovered in the 1940s, whereas daptomycin was discovered in the 1980s after screening an estimated 107 actinomycetes (PMID: 18524678).
Another common tactic is to grow organisms under different culture conditions and then test the growth media for bioactivity. Variables such as temperature, pH, composition of the growth medium, and the concentration of each component all influence secondary metabolite production in Actinomycetes. The presence or absence of another organism(s) in the same growth environment is yet another variable. Co-cultures involving two or more organisms might stimulate one of them to produce a compound not normally synthesized when they are grown as monocultures through secretion of key, uncharacterized signaling molecules or as a defensive mechanism. On the other hand, this approach suffers from several disadvantages that make successful implementation challenging. For example, it is nearly impossible to determine a priori the optimal growth environments that best stimulate production of different secondary metabolites, necessitating a large amount of trial and error. High-throughput miniaturized fermentation and screening methods mitigate but do not solve this problem since the number of different growth media is almost limitless. The use of co-cultures faces the same difficulty: the identity of appropriate helper strains, defined as organisms that stimulate others to produce bioactive molecules in co-culture, is not readily known.
A third method is to introduce random mutations into the genome of producer organisms. Random mutagenesis is a broad, well-established approach to microbial strain improvement; however, it also relies on the occurrence of a low probability event, the acquisition of one or more beneficial mutations, to succeed. Furthermore, while most random mutagenesis techniques such as UV irradiation, chemical mutagenesis, and error-prone PCR efficiently generate point mutations or small indels, they induce larger mutations such as large duplications, deletions, transpositions, or other genome rearrangements much less effectively. Conversely, mutational methods that focus on large genome rearrangements, such as whole genome shuffling, do not generate small point mutations efficiently. In this way, current random mutagenesis techniques only sample a small subset of all possible mutations even if multiple methods are utilized. An additional drawback is that strains usually become less fit as they acquire more mutations, a side-effect that can nullify their utility even though they might develop one or more beneficial mutations that confers a desired phenotype.
Targeted mutagenesis of key genes or pathways is a fourth method. This strategy is especially appealing for novel antibiotic production in Actinomycetes because the gene targets are well-defined: the PKS and NRPS clusters. Since these clusters contain distinct modules, their structural organization opens the possibility that different modules can be swapped among different clusters and among different Streptomyces spp., thereby potentially leading to numerous new molecules. The viability of this approach, referred to as combinatorial biosynthesis, has been demonstrated by the synthesis of 154 different hybrid PKS systems using individual modules from seven different PKS clusters in various streptomycetes and myxobacteria (PMID: 16116420 and 16187094). Each of the 154 hybrids contained two modules. The combinatorial biosynthesis of lipopeptide antibiotics related to daptomycin (PMID: 17090667) and spinosyn analogs (PMID: 17190446) are two other examples. Despite these successes, combinatorial biosynthesis has not led to an abundance of new bioactive molecules from Actinomycetes due to several technical challenges. For instance, the linker regions that connect one module to the next can vary within a given cluster and from one cluster to another, making it difficult to establish in a systematic way where one module ends and the next one begins. Moreover, swapping large pieces of protein-coding DNA inevitably impacts proper protein folding, frequently resulting in mis-folded proteins that are nonfunctional. More broadly, evidence is accumulating that mutations in other genes besides PKS and NRPS clusters also serve to improve existing antibiotic production or activate new ones (PMID: 20524642 and 19396160). Thus, a narrow focus on mutating only PKS and NRPS clusters could miss other important mutation sites in the genome.
The cloning and heterologous expression of antibiotic biosynthesis gene clusters in alternative host organisms is yet another method. This strategy is particularly attractive when no genetic manipulation system exists for the native producer, the sequence of interest comes from a metagenomic library, or a microorganism that cannot be cultured. These advantages are balanced by several disadvantages that limit the use of this tool for widespread antibiotic discovery. First, heterologous gene expression can lead to metabolic imbalances in the new host that then negatively impact the growth rate of the host or production of the new molecule. Second, cloning large stretches of DNA in streptomycetes is time consuming as the procedure still relies on classical methods using cosmids, fosmids, and similar constructs. Third, unknown but necessary cofactors, substrates or proteins might not be present in the new host.
The wealth of new antibiotics that undoubtedly remain to be discovered and the proven capability of existing techniques, such as those outlined above, to uncover new compounds argue for their continued implementation in the search for new antibiotics. At the same time, all existing techniques have drawbacks such that none constitute a single solution to the myriad of challenges faced during antibiotic discovery. As a result, there continues to be a need to develop new technologies and methods that complement and improve various aspects of the discovery process. This invention discloses a new method that allows for the discovery of novel, targeted antibiotics and, more generally, other types of bioactive compounds from a producing organism.